Grant 1--Integrated genetic, transcriptomic, and epigenetic analysis of cardiovascular disease phenotypes Progress: Analysis of datasets and construction of integrated networks was used to prioritize the top candidate HDL, LDL and triglyceride genes and identify the relevant mouse orthologs for subsequent mouse knockout studies. We have also used the integrated datasets to examine network relationships among various traits based on shared SNPs and DNA methylation patterns and preliminary analysis indicates novel trait associations involving coronary artery disease, alcoholism, cancer and hypertension. Efforts were devoted to further analysis and integration of various datasets in order to prioritize gene candidates having casual effects on lipid metabolism and then choosing mouse orthologs of the top candidates to be knocked out in mouse models. After a rigorous selection process we choose Abca6, Sidt2, and Aldh2 as the mouse genes to be tested. ABCA6 encodes a membrane-associated protein that is a member of the superfamily of ATP-binding cassette (ABC) transporters and is associated with LDL cholesterol levels; SIDT2 is associated with triglyceride levels in the human cohort; and ALDH2 is associated with HDL cholesterol levels in the human cohort. Generation of the relevant colony of individual mouse strains for subsequent knockout of the candidate genes is underway. Integration of datasets has allowed us to begin to identify novel associations among various pairs of traits based on shared SNPs and methylation patterns, including hypertension:cancer and coronary artery disease:alcoholism. Grant 2 Long-term effects of tobacco exposure on the methylomic signature Progress: We have completed analyses for an epigenome-wide association study of DNA methylation and cigarette smoking status. Our main result includes 18,760 statistically significant CpG sites annotated to 7,201 genes that are implicated in numerous vital pathways, such as signal transduction and transcription. In the past few months, we have also completed almost all the analyses needed to draft a manuscript on gene expression signatures associated with tobacco smoking. In brief, in the manuscript we will report a 1270-gene expression signature of smoking for current smoking, and a 39-gene expression signature for former smoking, meta-analyzing 12,000 samples from six cohort studies. The Levy Lab has completed a multidimensional project called the SABRe CVD Initiative that seeks to apply high throughput omic technologies to identify promising biomarkers of cardiovascular disease and its major risk factors. There are 4 sub-projects as part of the SABRe CVD Initiative. Project 1 Proteomics and Metabolomics SABRe CVD Project 1 has completed data collection to and now is in the analysis phase. The only lab work currently under way is replication studies based on the initial proteomics/metabolomics analyses. Available data resources generated by SABRe Project 1 are: 1. CVD sample set (336 case/control pairs (135MI C/C subset) total n=672) a. iTRAQ proteomics on 135 FHS MI case/control matched pairs b. Targeted LC/MS-MRM on 32 proteins on full CVD data set c. Targeted LC/MS Depletion-MRM on 28 proteins on full CVD data set 2. Metabolic Syndrome Factorial study set (n=652 samples) a. iTRAQ proteomics on 160 sample from factorial design study b. Targeted LC/MS-LIPDS MRM on 154 lipid species c. Targeted GC/MS on 155 metabolites on 670 individuals d. Replication of GC/MS metabolites in 650 samples from the BioImage study and replication of LC/MS metabolites in 650 samples from the PESA study On-going analysis: 1. The SABRe analysis team has published a proteomics paper titled Protein Biomarkers of New-Onset Cardiovascular Disease: A Prospective Study from the Systems Approach to Biomarker Research in Cardiovascular Disease (SABRe CVD) Initiative. As part of the SABRe CVD Initiative, we sought to identify plasma protein biomarkers of new-onset myocardial infarction (MI) and ASCVD in participants from the Framingham Heart Study (FHS). Identification of novel biomarkers that individually or aggregately predict risk of ASCVD could provide insight into the biology of the disease and could aid in developing targeted prevention strategies during the preclinical phase of ASCVD, when intervention may be more likely to alter disease progression. In this paper, in conjunction is SABRe Project 2, we report on the proteomics profiling analyses having identified single and multimarker protein panels that are associated with new onset ASCVD and may lead to a better understanding of underlying disease mechanisms. Our findings include many novel protein biomarkers that, if externally validated, may improve risk assessment for MI and ASCVD. 2. The SABRe analysis team has submitted a metabolomics paper titled Discovery and Independent Replication of Metabolomic Signatures of Metabolic Risk Factors. Here we report on the SABRe efforts that have identified multiple metabolomic biomarkers of metabolic risk factors including glutamic acid and other metabolites involved in the Krebs cycle whose levels are mediated by transaminase reactions. Understanding the pathways represented by our results may help unravel molecular derangements contributing to metabolic syndrome and its risk factors. 3. We have completed a draft publication of lipidomics LC/MS analysis of lipid biomarkers of metabolic risk factors. SABRe CVD Project 2: Targeted Protein Immunoassays 1. To date the contractor has completed measurements of 85 proteins in 17 different multiplex panels on over 7300 Framingham samples. All of these data sets are either available or in submission to NIH's dbGaP. On-going analysis: 1. The first 65 markers from SABRe project 2 were summarized in an abstract presented at the 2014 AHA Scientific Sessions. 2. An analysis pipeline exists to rapidly conduct analysis on each biomarker in association with all-cause mortality, CVD, and CHD on the new assay panels once they complete the data collection and QC stages. SABRe CVD Project 3: Gene Expression (mRNA) Analysis The SABRe CVD gene expression data resource created by Project 3 is completed and includes genome-wide transcriptomic profiling using the Affymetrix array 1.0 ST on approximately 5700 Framingham participant samples from the Offspring exam 8 and Third generation exam two cohorts. On-going analysis: 1. The SABRe Project 3 analysis team is coordinating several different data analysis projects using the data set. The table below includes some of the various research projects utilizing the data resources. They are at varying points in their analysis plans. SABRe CVD Project 4-miRNA Profiling The data resource for SABRe CVD Project 4 is also completed. We completed measurement of 350 miRNA across 5700 Offspring and Third Generation cohort participant samples. All data are available in dbGaP. On-going analysis: 1. The SABRe Project 4 analysis team has several cross over projects with the Project 3 team as these compromise the systems biology approaches of SABRe. The table below is some of the SABRe Research proposals making use of the Project 4 data resource.